Anaplerotic therapy of huntington disease and other polyglutamine diseases

ABSTRACT

The present invention relates to a method for treating and/or preventing Huntington disease and other polyglutamine diseases, comprising the step of administering an effective amount of a precursor of propionyl-CoA to an individual in need thereof.

This application is a Continuation of U.S. patent application Ser. No.14/308,966, filed Jun. 19, 2014, which issued as U.S. Pat. No.9,717,705, which is a Continuation of U.S. patent application Ser. No.13/341,028, filed Dec. 30, 2011, which is a Continuation of U.S. patentapplication Ser. No. 12/516,486, filed May 27, 2009, which was filedpursuant to 35 U.S.C. 371 as a U.S. National Phase Application ofInternational Patent Application No. PCT/EP07/63181, filed Dec. 3, 2007,claiming the benefit of priority to European Patent Application No.06291873.5, filed Dec. 4, 2006. The entire text of the aforementionedapplications is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the treatment and the prevention ofHuntington disease and other polyglutamine diseases.

BACKGROUND OF THE INVENTION

Huntington disease (HD) is a devastating inherited neurodegenerativedisease without curative treatment. HD is the founding member of a largegroup of diseases due to polyglutamine accumulation and toxicity. Thereis a critical need for new insights in the pathophysiology of thisdisease, as well as for the identification of relevant molecules forclinical trials.

Several observations have led to the hypothesis that mitochondrialdysfunction has a role in polyglutamine diseases, and in Huntingtondisease in particular. Several lines of evidence indicate abnormalenergy metabolism, including reduced glucose metabolism, elevatedlactate levels and impaired mitochondrial-complex activity (Di Prospersand Fischbeck 2005, Nat Rev Genet 6(10): 756-65). To explain thisabnormal energy metabolism most studies favoured a secondary impairmentof the mitochondrial respiratory chain. An important decrease incomplexes II & III (55%) has been shown in the caudate of HD patients(Gu 1996, Ann Neurol 39: 385-9), as well as a deficiency in complex 1 inmuscle (Arenas 1998, Ann Neurol 43:397-400), therefore supporting thepossibility of mitochondrial respiratory chain defects in pathogenesisof HD (Shapira 1998, Biochem Biophys Acta 1366: 225-33, Grunewald 1999,Ann N Y Acad Sci 893: 203-13). These findings correlated with HD modelsinduced by 3-nitropropionic acid, an irreversible complex II inhibitor(Beal 1993, J Neurosci 13: 4181-92). However, these data arecontroverted by the demonstration of normal mitochondrial electrontransport complexes in transgenic mice at an early stage (Guidetty 2001,Exp Neurol 169: 340-50), as well as in striatal cells in cultureexpressing mutant huntingtin, despite the significant reduction in ATPsynthesis observed in those cells (Milakovic 2005, JBC 280: 30773-82).Additional indirect evidence for an energy defect in polyglutaminedisease arise from the partial efficacy of energetic therapies, such asdichloroacetate (Andreassen 2001, Ann Neurol 50: 112-9), pyruvate (Ryu2004, Exp Neurol 187: 150-9), creatine (Ferrante 2000, J Neurosci 20:4389-97) and coenzyme Q10 (Schilling 2001, Neurosci Lett 315: 149-53) inmice models.

However, to date, effective pharmacotherapy for neurodegenerativedisease associated with impaired energy metabolism like polyglutaminediseases in particular, remains rather elusive.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a methodfor treating and/or preventing polyglutamine disease such as Huntingtondisease.

In fulfilling this object, there is provided a method for treatingand/or preventing a polyglutamine disease, comprising the step ofadministering an effective amount of a precursor of propionyl-CoA to anindividual in need thereof.

Also provided is the use of a precursor of propionyl-CoA in themanufacture of a medicament for treating and/or preventing polyglutaminedisease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating and/or preventing apolyglutamine disease, comprising the step of administering an effectiveamount of a precursor of propionyl-CoA to an individual in need thereof.

By individual, it is meant animal or human being.

Also provided is a precursor of propionyl-CoA for treating and/orpreventing a polyglutamine disease.

Also provided is the use of a precursor of propionyl-CoA in themanufacture of a medicament and/or a food substance for treating and/orpreventing a polyglutamine disease.

Polyglutamine diseases constitute a class of nine genetically distinctdisorders that are caused by expansion of translated CAG repeat. Theseinclude Huntington disease (HD), dentatorubralpallidoluysian atrophy(DRPLA), spinal and bulbar muscular atrophy (SBMA) and spinocerebellarataxia 1, 2, 3, 6, 7 and 17. Although the disease causing proteins areexpressed widely in the central nervous system, specific populations ofneurons are vulnerable in each disease, resulting in characteristicpatterns of neurodegeneration and clinical features.

In an embodiment of the present invention, the polyglutamine disease isselected from the group consisting of Huntington disease (HD),dentatorubralpallidoluysiam atrophy (DRPLA), spinal and bulbar muscularatrophy (SBMA), spinocerebellar ataxia 1, 2, 3, 6, 7 and 17.

In a preferred embodiment of the present invention, the polyglutaminedisease is Huntington disease.

By precursor of propionyl-CoA, it is meant a substance from whichpropionyl-CoA can be formed by one or more metabolic reactions takingplace within the body. Examples of precursors of propionyl-CoA are shownin FIG. 5. Typical examples of precursors of propionyl-CoA areodd-medium-chain fatty acids, in particular seven-carbon fatty acid,triheptanoin (triheptanoyl-glycerol), heptanoate, C5 ketone bodies (e.g.β-ketopentanoate (3-ketovalerate), and β-hydroxypentanoate(3-hydroxyvalerate)) (Kinman 2006, Am J Physiol Endocrinol Metab 291(4): E860-6, Brunengraber and Roe 2006. J Inherit Metabol Dis 29 (2-3):327-31).

The examples of precursors of propionyl-CoA described above include thecompounds themselves, as well as their salts, prodrugs, solvates, ifapplicable. Examples of prodrugs include esters, oligomers ofhydroxyalkanoate such as oligo(3-hydroxyvalerate) (Seebach 1999, Int JBiol Macromol 25 (1-3): 217-36) and other pharmaceutically acceptablederivatives, which, upon administration to a individual, are capable ofproviding propionyl-CoA. A solvate refers to a complex, formed between,a precursor of propionyl-CoA described above and a pharmaceuticallyacceptable solvent. Examples of pharmaceutically acceptable solventsinclude water, ethanol, isopropanol, ethyl acetate, acetic acid, andethanolamine.

A very practical dietary source of propionyl-CoA is triheptanoin(triheptanoyl-glycerol). After intestinal hydrolysis of triheptanoin,heptanoate is absorbed in the portal vein. In the liver, it is partiallyconverted to the C5 ketone bodies β-ketopentanoate (3-ketovalerate), andβ-hydroxypentanoate (3-hydroxyvalerate). The C5-ketones bodies are alsoprecursors of propionyl-CoA in peripheral tissues. Thus after ingestionof triheptanoin, peripheral tissues receive two precursors ofpropionyl-CoA, i.e., heptanoate and C5-ketone bodies. Quiteinterestingly, C5-, like C4-, ketone bodies are natural substrates forthe brain and can target physiological receptors at the surface membraneof the blood brain barrier. The demonstration of the transport ofC5-ketone bodies across the blood-brain barrier was recently provided bythe treatment of a patient with pyruvate carboxylase deficiency, wherecerebral anaplerosis is primarily impaired (Mochel 2005, Mol Genet Metab84: 305-12). The availability of C5-ketone bodies for cerebralanaplerosis was also demonstrated by the normalisation of glutamine andGABA in the CSF of this patient, as well as the absence of brainpathology.

The person skilled in the art is aware of standard methods forproduction of precursors of propionyl-CoA.

In a preferred embodiment of the present invention, the precursor ofpropionyl-CoA is triheptanoin, heptanoic acid or heptanoate.

Triheptanoin has already been used in the anaplerotic treatment of a fewpathologies having in common a decrease in ATP production in spite ofample supply of acetyl-CoA to the citric acid cycle (CAC), and a normalrespiratory chain. Such pathologies include cardiac reperfusion injury(Reszko 2003, JBC 278: 34959-65), long-chain fatty acid oxidationdisorders (FOD) (Roe 2002, JCI 110: 259-69 and WO 0045649), pyruvatecarboxylase deficiency (Mochel 2005, Mol Genet Metab 84: 305-12) andglycogen storage disease type II (Roe and Mochel 2000, J Inherit MetabDis 29 (2-3): 332-40)

Triheptanoin is a triglyceride made by the esterification of threen-heptanoic acid molecules and glycerol. In regard to therapy, the termsheptanoic acid, heptanoate, and triheptanoin may be used interchangeablyin the following description. Also, it will be understood by one skilledin the art that heptanoic acid, heptanoate, and triheptanoin areexemplary precursors of propionyl-CoA of the invention. Substituted,unsaturated, or branched heptanoate, as well as other modifiedseven-carbon fatty acids can be used without departing from the scope ofthe invention.

Heptanoic acid is found in various fusel oils in appreciable amounts andcan be extracted by any means known in the art. It can also besynthesized by oxidation of heptaldehyde with potassium permanganate indilute sulfuric acid (Ruhoff Org Syn ColI. volII, 315 (1943)). Heptanoicacid is also commercially available through Sigma Chemical Co. (St.Louis, Mo.).

Triheptanoin can be obtained by the esterification of heptanoic acid andglycerol by any means known in the art. Triheptanoin is alsocommercially available through CondeaChemie GmbH (Witten, Germany) asSpecial Oil 107.

Unsaturated heptanoate can also be milked in the present invention. Inaddition, substituted, unsaturated, and/or branched seven-carbon fattyacids which readily enter the mitochondrion without special transport,enzymes can be utilized in the present invention. For example,4-methylhexanoate, 4-methylhexanoate, and 3-hydroxy-4-methylhexanoateare broken down by normal b-oxidation to 2-methylbutyric acid with finaldegradation accomplished via the isoleucine pathway. Likewise,5-methylhexanoate, 5-methylhexanoate, and hydroxy-5-methylhexanoate arebroken down by normal b-oxidation to isovaleric acid with funddegradation accomplished via the leucine pathway.

Precursors of propionyl-CoA of the present invention can be administeredorally, parenterally, or intraperitoneally. Preferably, it can beadministered via ingestion of a food substance containing a precursor ofpropionyl-CoA such as triheptanoin at a concentration effective toachieve therapeutic levels. Alternatively, it can be administered as acapsule or entrapped in liposomes, in solution or suspension, alone orin combination with other nutrients, additional sweetening and/orflavoring agents. Capsules and tablets can be coated with sugar, shellacand other enteric agents as is known. Typically medicaments according tothe invention comprise a precursor of propionyl-CoA, together with apharmaceutically-acceptable carrier. A person skilled in the art will beaware of suitable carriers. Suitable formulations for administration byany desired route may be prepared by standard, methods, for example byreference to well-known text such as Remington: The Science and Practiceof Pharmacy.

In the following, the invention will be illustrated by means of thefollowing examples as well as the figures.

FIGURE LEGENDS

FIG. 1: Partial least square (PLS) analyses of NMR spectra of plasmasamples from HD patients with no or little signs of the disease andcontrols. Three groups of premanifest, early and mildly affected HDpatients were constituted on the basis of their UHDRS scores, asdescribed in the methods. The first and second components in the X space(NMR spectrum) are denoted PC[1] and PC[2] respectively. PLS score plots(PC[1]/PC[2]) of pair-wise compared groups show the greater variationwithin the NMR spectrum according to a priori classification with UHDRS.There is a clear separation between premanifest and early HD patients(a), as well as between early and mildly affected HD patients (b).Therefore, plasma NMR spectroscopy allows separation of HD patients atdifferent stages of the disease. Despite some overlap, differentiationbetween controls and premanifest individuals is also observed (c).

FIG. 2: Plasma relative concentrations of branched chain amino acids areresponsible for separation between HD groups. PLS-contribution plotallows comparison between plasma metabolic profiles from early affectedHD patients to premanifest carriers. NMR variables that have thegreatest weight (w*₁, sealed in units of standard deviation), thereforecontributing most to the separation between HD groups, are: decreasedconcentrations (>2SD) of metabolites located between 0.9 and 1.05 ppm:valine, leucine and isoleucine. The same contribution plot was obtainedwhen comparing-plasma metabolic profiles from mildly to early affectedHD patients (data not shown).

FIG. 3: The levels of branched chain amino acids are significantlydifferent in HD patients and controls. The concentrations of valine,leucine and isoleucine in plasma were determined by ion exchangechromatography. Comparisons of means (ANOVA) were made between men orwomen with HD and their respective controls. In men, there is asignificant decrease of valine, leucine and isoleucine in the HD group.In women, similar results are observed for leucine and isoleucine. Ofnote, in both men and women, the comparison of the standard deviationsof valine, leucine and isoleucine values shows almost no overlap betweenthe control and the HD groups.

FIG. 4: Plasma branched chain amino acids are negatively correlated withdisease progression in HD. Principal component analysis (PCA) loadingplot shows the relative importance of each variable from the study andthe correlation between these variables. The more the loading (p) ofeach variable diverges from zero, the more this variable is important inthe explained variance of the given component (expressed by R2x). Theexplained variance of all data reach 44% in the first component and 22%in the second component. There is strong negative correlation betweenclinical markers (the size of the abnormal CAG repeat expansion, diseaseseverity measured by the UHDRS and depression scores) and parametersassociated with weight (weight, BMI, LBM and FBM for lean and fat bodymass respectively). The BCAA, valine, leucine and isoleucine, arenegatively correlated with disease progression and positively correlatedwith weight loss. Note that the number of CAG repeats are negativelycorrelated with BCAA values: the larger the repeat the lower the BCAAvalues (p=0.015 for valine, 0.018 for leucine and 0.020 for isoleucine).

FIG. 5: Diagram depicting the metabolic pathway of triheptanoin.

FIG. 6: Purkinje cell survival 12 days and 20 days following infectionwith the lentiviral vectors. 100Q: ATXN7T-100Q-GFP. 10Q: ATXN7T-10Q-GFP,GFP: control vector expressing GFP alone. The Purkinje cell survival isexpressed as the percentage of Calbindin-positive cells in infectedcultures compared to non-infected cultures, 12 days after infection byATXN7T-100Q-GFP, Purkinje cell survival is reduced to ˜30% and fartherdecreases to ˜15% after 20 days demonstrating the high and progressiveneurotoxicity of the mutant protein, which is clearly distinct from thetoxicity of the viral vector alone (GFP condition).

FIG. 7: Weight and food, intake evolution of female knock-in mice (n=3)and female wild-type mice (n=3) from 7 to 11 weeks of age.

EXAMPLES

In the following description, all molecular biology experiments forwhich no detailed protocol is given are performed according to standardprotocol.

Example 1: Identification of a Plasma Biomarker in Premanifest Carriersof Huntington Disease Indicating Early Energy Unbalance

Abbreviations: HD (Huntington disease), UHDRS (Unified Huntingtondisease rating scale), ppm (parts per million), PCA (principalcomponents analysis), PLS (partial least square).

Abstract

Huntington disease (HD) is an autosomal dominant neurodegenerativedisorder in which an energy deficiency is thought to play a role.Patients consistently lose weight, although the reason for this isunknown. In view of the specific access to premanifest carriers in HD,we performed a multiparametric study in a group of 32 individuals withno sign or little of the disease compared to 21 controls. Weight losswas observed even in premanifest carriers in the HD group, althoughtheir calorie intake was higher. Inflammatory processes were ruled out,as well as primary hormonal dysfunction, including ghrelin and leptinbalance. Proton nuclear magnetic resonance spectroscopy on plasma did,however, distinguish HD patients at different stages of the disease andpremanifest carriers from controls. Differences between groups wereattributable to low levels of the branched chain amino acids (BCAA),valine, leucine and isoleucine. We confirmed that BCAA levels werenegatively correlated with weight loss and more importantly with diseaseprogression. Levels of insulin growth factor type 1 (IGF1), which isregulated, by BCAA, were also lower in the HD group than in controls.BCAA are, therefore, the first biomarkers identified in HD and offer newinsights into an underlying early energy deficiency.

Results

Evidence of Early Hypercatabolism in HD

Three groups were defined according to their UHDRS scores. There were 15carriers of the mutation without any motor or cognitive signs of HD(UHDRS 0.5±1.0), 10 patients in an early stage of the disease (UHDRS11.9±4.9) and 7 patients mildly affected (UHDRS 44.4±14.1)

Weight loss during the last 5 years was significantly greater in the HDgroup than in controls (p<0.0001). The difference remained significantwhen men (p=0.002) and women (p=0.003) were analyzed separately. Inspite of higher intake of calories, HD individuals and controls showedno different BMI. Importantly, in HD men, BMI was significantly lowerthan in controls, and total calories were even inversely correlated withweight (p=0.029) and lean body mass (p=0.004).

These observations confirm that weight balance is abnormal early in HD.Even in premanifest carriers, the nutritional pattern of HD patientsdiffered from that of controls; they had significantly higher caloricintake (2195±495, n=15 versus 1665±305, n=21, p<0.001) and greaterprotein intake (85±24, n=15 versus 70±14, n=21, p=0.025). Theseobservations clearly show hypercatabolism in the HD group, even in thevery early stages of the disease. Relationship to the disease was notexplained by common, causes of hypercatabolism such as inflammation orclassical endocrine dysfunctions. Indeed, ERS, CRP, serum interleukins1ß and 6 and serum fasting cortisol, T4L and TSH were similar in the HDand control groups. There was no glycosuria, and fasting blood glucoseand insulin levels were in the normal range is the HD group.

Identification of Candidate Biomarkers by Plasma ¹H NMR Spectroscopy(FIGS. 1 and 2)

PCA on plasma NMR spectra, identified no outliers in both the controland HD dataset. PLS analyses could distinguish HD individuals atdifferent stages of the disease, meaning that underlying plasmametabolites behaved differently. The difference between premanifestcarriers and early HD was evident (FIG. 1a ), and extended to moreadvanced stages of the disease (FIG. 1b ). In addition, controls andpremanifest carriers did not have the same metabolic profile, despitesome overlap (FIG. 1c ).

The spectral region that contributed to differences among the HD groupsdetermined from PLS contribution plots is show in FIG. 2. Plasmametabolic profile from early affected HD patients to premanifestcarriers was compared, as well as from mildly to early affected HDpatients. There was a significant (>2SD) decrease along with diseaseprogression in the plasma concentrations of a group of variables fromthe buckets located between 0.9 to 1.05 ppm on the NMR spectrum. Thesepeaks correspond to the branched chain amino acids (BCAA), valine,leucine and isoleucine. No other significant differences among thegroups were detected in the spectra even though very small buckets (0.02ppm) were analyzed. This indicates that a selective decrease in BCAAconcentrations accompanies the progression of the disease, and evendistinguishes premanifest carriers from both controls and early HDpatients. Plasma BCAA levels appear, therefore, to be relevantbiomarkers of HD.

Confirmation that BCAA are Affected in HD (FIGS. 3 and 4)

To confirm that BCAA are affected in HD, we also measured theirconcentrations in plasma by ion exchange chromatography. Valine, leucineand isoleucine levels were significantly lower in the HD group comparedto controls (p=0.009, p<0.001 and p=0.002, respectively). In addition,the levels of each BCAA were significantly correlated with the observedweight loss in the patients (p=0.005, 0.002 and 0.014 respectively).More importantly, BCAA levels were negatively correlated with UHDRSvalues (p=0.017, <0.0001 and 0.003 respectively) in both men (p=0.035,0.019 and 0.036 respectively and women (p=0.007 for leucine and 0.01 forisoleucine). Although the BMI values of women with HD were similar tothe controls, they had significantly lower leucine (p=0.002) andisoleucine (p=0.014) levels (FIG. 3). This indicates that lower BCAAlevels are not only associated with weight balance, but more importantlywith Huntington disease itself, interestingly, the plasma levels of thethree BCAA were significantly lower in patients at an early stage of thedisease compared to premanifest carriers (p=0.042, 0.019 and 0.024respectively). BCAA levels are, therefore, associated with diseaseonset, emphasizing that they can be used as reliable biomarkers in HD.When comparing premanifest carriers to controls, the plasma levels ofvaline (228±50 versus 245±44), leucine (130±24 versus 144±23) andisoleucine (62±12 versus 68±15) were lower m the former group, althoughnot significantly. This is likely due to the heterogeneity of thepremanifest group in which the estimated time to disease onset isexpected to vary between individuals, so that the metabolic profile ofsome premanifest carriers can be similar to controls.

A multivariate PCA confirms that there is a strong negative correlationbetween clinical markers (abnormal CAG repeat expansion, size, UHDRSscores, depression scores) and weight parameters (FIG. 4). Low BCAAvalues appear to be the strongest variables that are negativelycorrelated with HD and positively correlated with weight loss. Thenumbers of CAG repeats are also negatively correlated with BCAA values:the larger the repeat the lower the values (p=0.015 for valine, 0.018for leucine and 0.020 for isoleucine).

The other metabolic markers (serum cholesterol and triglycerides,remaining amino acids and acylcarnitines in plasma, organic acids inurine) were similar in the HD group and in controls. IGF1 levels,however, were significantly lower in the HD group (p=0.0011) andnegatively correlated with UHDRS scores (p=0.004). IGF1 levels were alsocorrelated with leucine (p=0.04) and isoleucine (p=0.02) levels, whichwas expected since IGF1 is regulated by BCAA. However, this has to bemodulated by the fact that IGF1 levels are known to decrease with age,as observed in our HD cohort (p=0.002). The decrease in IGF1 levels wasnot associated with significant changes in other nutritional parameters(albumin, prealbumin, orosomucoid). There was no correlation eitherbetween IGF1 levels and parameters associated with weight (BMI, lean andfat body mass) or food intake.

Discussion

This is the first extended investigation of weight disorder in HD. Wehave shown that weight loss begins early in the disease, despite highercalorie intake, and is evident even in premanifest mutation carriers andthose with little or no chorea. This hypercatabolism cannot be explainedby common mechanisms like inflammation or altered endocrine functions,both of which have been incriminated in the pathophysiology of HD(Kremer et al. 1989; Pavese et al. 2006). Hypercatabolism is HD seems,therefore, to be part of the pathological process induced by thedisease. Our ¹H NMR spectroscopy analysis shows that patients atdifferent stages of HD can be distinguished from each other, andpremanifest mutation carriers can be distinguished from controls, on thebasis of their plasma, levels of branched chain amino acids. Thedecrease in the levels of these amino acids correlated with weight lossin HD patients, but more importantly with the severity of the clinicalimpairment, i.e., with Huntington disease itself. This finding issupported by previous studies in which a decrease in plasma BCAA wasdocumented in more severely affected HD patients (Perry et al. 1969;Phillipson and Bird 1977; Reilmann et al. 1995). The extensive metabolicscreening we performed in combination with an independent techniqueconfirmed that plasma BCAA were the only metabolites that differedbetween the HD group and controls. This difference existed regardless ofsex and between patients at an early stage of the disease andpremanifest carriers. Consequently, plasma BCAA can be considered asrelevant biomarkers for Huntington disease. They should help to detectthe onset of the disease and to monitor its progression in view oftherapeutic trials. To our knowledge, this is the first accessiblebiomarker identified in Huntington disease and, more widely, the firstperipheral biomarker evidenced in a neurodegenerative disorder.

Only few metabonomic studies have led to the identification ofbiomarkers that can be used routinely for the follow up of patients(Sabatine et al. 2005). Inter-individual variability is known tocomplicate such analyses in human body fluids, thus restrictingmetabonomic studies essentially to animal models (Wagner et al. 2006).Common experimental and analytical biases in humans include dietaryintake, time of sample collection, sample conditioning and chemicalshifts due to changes in pH (Cloarec et al. 2005; Teahan et al. 2006:Walsh et al. 2006). In the present study, each of these parameters wasrigorously controlled. This probably explains the accuracy of our NMRfindings.

The implication of BCAA in mitochondrial intermediary metabolism, bothin brain and peripheral tissues, further supports an important role forenergy deficit in HD. A reduction in ATP production was shown in brainof HD mice, including presymptomatic mice (Gines et al. 2003). Asignificant reduction in ATP levels and mitochondrial respiration wasalso evidenced in striatal cells of HD mice, although the respiratorychain complexes were not impaired (Milakovic and Johnson 2005). In HDpatients, there is strong evidence for hypermetabolism in the brainwhere glucose consumption is reduced, especially in the basal ganglia,even in presymptomatic mutation carriers (Grafton et al, 1992; Kuwert etal. 1993; Antonini et al. 1996). The underlying cause of this earlyenergy deficit in HD brain is currently unknown, but impaired glycolysis(Browne and Beal 2004), citric acid cycle (Tabrizi et al. 1996). Theand/or oxidative phosphorylation (Milakovic and Johnson 2005) may beinvolved. Recently, mutated huntingtin was shown to decrease theexpression, of PGC-1α (peroxisome proliferators-activated receptor gammacoactivator-1α) in the striatum of HD mice and patients, through aCREB-dependent transcriptional inhibition (Cui et al. 2006). PGC-1α is atranscriptional coactivator that regulates key energetic metabolicpathways, both in the brain and peripheral tissues (Lin et al. 2005).The possible role of PGC-1α in HD was initially suspected from theobservation of selective striatal lesions in the PGC-1α knockout mouse(Lin et al. 2004). Down-regulation of PGC-1α in HD striatum was thenshown to affect mitochondrial energy metabolism, possibly by impairingoxidative phosphorylation (Cui et al. 2006). In addition, the inhibitionof succinate dehydrogenase, by 3-nitropropionic acid or malonate, mimicsHD neuropathology in mice (Klivenyi et al. 2004), indicating that a lackof substrates for the citric acid cycle and the respiratory chain isimplicated in the energy deficit in HD brain. Importantly, mitochondrialoxidation of BCAA leads to the production of acetyl-CoA andsuccinyl-CoA, two key intermediates of the citric acid cycle.Insufficient caloric or protein intake was excluded in our study, aswell as impairment of the BCAA oxidation pathway since organic acidlevels in urine were normal. Therefore, the decrease in plasma BCAAobserved in the HD group probably results from the activation of acompensatory mechanism to provide energy substrates to the citric acidcycle, as described in various cachexia-producing illnesses (Szpetnar etal. 2004; De Bandt and Cynober 2006). The correlation between decreasedBCAA levels and weight loss in our study suggests that excessivemobilization and oxidation of BCAA to produce energy in muscle isassociated with weight loss and reduced lean body mass. The observationof weight loss prior to neurocognitive decline suggests thatneurological symptoms are exacerbated when substrates from the peripherybecome insufficient to compensate for the energy deficit in the HDbrain. The normal rate of oxygen consumption recently observed afterstriatal infusion of succinate (Weydt et al. 2006) supports the ideathat providing energy through an increase in both systemic and cerebralcitric acid cycle intermediates may be a promising therapeutic approachin HD.

On the pathophysiological level, our study also showed that low plasmaBCAA levels result in low IGF1 levels in the HD group compared tocontrols although they have a higher protein and caloric intake. Thetight connection between IGF1 and essential amino acids has beenextensively studied (Straus and Takemoto 1988; Harp et al. 1991; Thissenet al. 1994; Gomez-Merino et al. 2004). The availability of essentialamino acids seems to have a greater effect on IGF1 gene expression thanhormonal stimuli such as serum insulin concentrations (Master et al.1989), IGF1 is also a more sensitive indicator of nutrient repletionthan albumin, prealbumin or orosomucoide, as observed in our study.Interestingly, huntingtin is a substrate of the serine-threonine Aktpathway, which is activated by IGF1 (Humbert et al. 2002). Alteredactivation of the Akt pathway has been shown to decrease phosphorylationof the mutated huntingtin, resulting in an increased neuronal toxicity(Rangone et al. 2005). Low levels of IGF1 in HD parents might thereforeprovide an explanation of the alteration in Akt activation observed inHD cellular models. Consequently, increasing BCAA levels to correct thedeficit in IGF1, should favor the phosphorylation of mutated huntingtin,thereby decreasing its toxicity.

In conclusion, the combination of a rigorous nutritional assessment andmetabonomic tools has provided new insight into HD. We have demonstratedthe existence of an early energy deficiency in this neurodegenerativedisorder, reflected by weight loss. We have also identified the firstreliable biomarker in Huntington disease. The evidence of decreasedplasma BCAA levels in very early stages of HD highlights the possibilityfor therapies aimed at supplying a sufficient pool of acetyl-CoA tocompensate for the early energy deficit.

Altogether, these data are supportive of a causative role for energydeficiency in Huntington's disease. Rather than a defect in therespiratory chain, low plasma BCAA levels indicate an energeticimpairment in the Krebs cycle. Therefore, molecules selected for theirability to reverse this deficiency in mitochondrial ATP productionshould refill the pool of catalytic intermediates of the Krebs cycle.Dietary compounds such as triheptanoin have recently been used in humantherapeutic trials for their ability to refill the pools of catalyticintermediates of the Kreb's cycle, a key energetic process calledanaplerosis. Because of additional evidence for triheptanoin metabolitesto cross the blood brain barrier, anaplerotic therapies representpromising molecules for reversing the energy deficiency associated withneurodegenerative diseases and thereby correcting some if not allclinical manifestations of these diseases.

Material and Methods

HD Patients, Premanifest Carriers and Controls

We included 32 individuals with abnormal CAG repeals expansions (>36) inthe HD1 gene (19 women and 13 men) and 21 controls (13 women and 8 men).In the HD group, 15 were premanifest carriers who had before applied forpredictive testing due to their risk for HD, 10 were at a very earlystage of the disease and 7 had moderate signs of the disease. Controlswere healthy volunteers in the same age range, unrelated to HDindividuals. All participants were examined and blood samples were takenduring a single visit to the reference centre for HD at the SalpëtrièreHospital (Paris). Patients, premanifest carriers and controls wereenrolled in a clinical protocol authorized by the Assistance Publiquedes Hôpítaux de Paris (CRC 05129), and approved by the local ethicscommittee. Informed consent was obtained for all participants.

Determination of Weight Balance and Food Intake

Height and weight were recorded the day of the clinical, examination.Weight loss was calculated by subtracting current weight from the weightof the patient 5 years before inclusion in the study. This informationwas obtained daring the interview and was verified retrospectively fromthe patients' medical files. The body mass index (BMI) was obtained bydividing weight (in kilograms) by height (in meters) squared.Bioelectrical impedance (Tanita®) was measured to evaluate the lean massand fat mass of all participants (Segal et al. 1988).

To determine food intake, HD patients at early stages, premanifestcarriers and controls prospectively recorded their normal foodconsumption during 3 days preceding their examination. The accuracy ofthe 3-days record was verified one month later with a questionnaireassessing food intake over a 24-hours period that was sent to the homesof all participants. A professional dietician (CG) used these twodocuments to calculate mean total calories, and protein, lipid and sugarintake for both the HD and control groups using an automated system(Diaeta Software®).

Multiparametric Evaluation of Weight Balance

A standardized protocol was designed to thoroughly evaluate all possiblecauses of weight loss and to avoid biases related to food intake andcircadian changes. It included sequentially: (i) a minimal 12 hours lastthe night preceding the examination, (ii) and morning blood and urinecollection at the same hour (9 am) Samples were stored on ice forimmediate analyses or frozen at −80° C. for further analyses.

Standard analyses included blood cell count, blood and urine glucose,serum electrolytes, and basic nutritional parameter, such as serumcholesterol triglycerides, albumin, prealbumin and orosomucoid. Torefine the evaluation of nutrient repletion, serum insulin growth factortype 1 (IGF1) concentrations were measured using a specificimmunoradiometric assay (IGF1 RIACT, Cis-Bio International,Gif-sur-Yvette, France). The three main axes involved in the regulationof weight balance were explored: inflammation, endocrine function andintermediary metabolism. The evaluation of inflammation includeddetermination of the erythrocyte sedimentation rate (ESR) andquantification of C-reaetive protein (CRP) and the serum interleukinsIL1β and IL6 by ELISA (Diaclone, Besançon, France). Besides serum IGF1,the basic endocrine evaluation included measurements of fasting serumcortisol (at 9 am), tetraiodothyronine (T4L), thyroid stimulating,hormone (TSH) and insulin (Elisa Access ultrasensitive insulin, BeekmanCoulter, Roissy, France).

We explored intermediary metabolism through analysis of (i) plasma aminoacids using ion exchange chromatography alter coloration by ninhydrine(Aminotag, Geol), (ii) organic acids in urine by gas chromatography (GSVariant 3400) coupled to mass spectrometry (Ion trap, Saturn 2000,Variant) after extraction with ethyl acetate and derivation bysilylation, (iii) the plasma acylcarnitines nines profile by tandem massspectrometry (Applied Biosystem) with electrospray ionization (ESI) andFIA (flow injection analysis). Acylcarnitines were identified by using aprecursor ion m/z 85 scan and quantified in MRM (multiple reactionmonitoring) mode. Acetylcarnitine (C2-carnitine) levels were used tosurvey the lasting status of both HD individuals and controls (Costa etal. 1999).

¹H Nuclear Magnetic Resonance Spectroscopy (NMR) on Plasma

Plasma samples were prepared for ¹H NMR spectroscopy with minimalhandling. Plasma samples were deproteinized using a 10 kDa filter(Nanosep, Omega) to avoid interference from high molecular weightspecies such as lipoproteins. Before use, the filter was washed twicewith water by centrifugation to remove glycerol. A 100 μl aliquot of3.89 mM [trimethylsilyl]-2,2,3,3-tetradeuteropropionic acid in ²H₂O(TSP-²H₂O, Aldrich) was added to 500 μl of the ultrafiltrate, providinga chemical shift reference (δ=0.00 ppm), a concentration reference and adeuterium lock signal. The pH of the ultrafiltrate was adjusted to2.50±0.05 with concentrated HCl. Finally, 500 μl of the sample wasplaced in a 5 mm NMR tube (Wilmad Royal Imperial). The ¹H NMR spectrawere determined on an Avance-500 SB spectrometer (Bruker, France)equipped with a 5 mm BBI (broadband inverse) probe; samples were notspun. Spectra were collected at 25° C. and consisted in 32K data pointswith a spectral width of 6,000 Hz and a total acquisition times of 27min. A 90° radiofrequency pulse, following a water signal presaturationof 10 s, was used for each 128 scans. Shimming of the sample wasperformed automatically on the deuterium signal. The resonance linewidths for TSP and metabolites were <1 Hz. Before a Fouriertransformation into 64K data points, a sine-bell squared filter (SSB=2)was used to reduce noise. The phase and the baseline were correctedmanually using the spectrometer software (X-Win NMR 3.5, Bruker,France). NMR spectra were first analyzed individually in order to detectabnormal signals—i.e. treatment or special food—that could furtherinterfere with global analyses. For statistical analyses, spectra weredata reduced in numerical format by integrating spectral regions(buckets) every 0.02 ppm and scaled to the total intensity of thespectrum with Amix 3.6.8 software (Bruker Analytische Messtechnik,Germany) from 0.8 to 8.6 ppm, the water peak area being excluded fromeach spectrum (4.4 to 5.2 ppm). Accordingly, each bucket from the NMRspectrum corresponded to a single variable.

Statistical Analyses

Metaboonomic studies consist in multivariate statistical analyses, e.g.principal components analysis (PCA) and partial least squares analysis(PLS), with as many components as variables. Multivariate analyses ofthe data obtained by NMR spectroscopy were performed with Simca-P® 11.0software (Umetrics, Sweden). For PCA and PLS, unit variance scaled datawere used to ensure the inclusion of metabolites present in both highand low concentrations. Each variable was mean centered and computed as1/SD_(j), standard deviation of variable j computed around the mean. PCAconsiders each bucket from the NMR spectrum as an X variable and wastherefore used to discern the presence of inherent similarities betweenspectral profiles and to identity outliers. PLS is a regressionextension of PCA and best describe the variation within the dataaccording to a priori classification, corresponding to a Y variable,which was the UHDRS score in our study. PLS was used to identifyprincipal components maximizing the covariance between all X (NMRspectrum) and Y (UHDRS) variables. The greatest dispersion of thespectral profiles is usually best observed in the two first componentsof the analyses. The first and second components in the X space (NMRspectrum) were denoted PC[1] and PC[2] respectively. Therefore, PLSscore plot (PC[1]/PC[2]) of pair-wise compared groups displayed thegreater variation within the NMR spectrum according to UHDRS. Thevalidity of each component was obtained by cross validation.Contribution plot was then analyzed in order to determine the respectiveweight of variables contributing most to the separation between groups.

For comparison of means, ANOVA or non-parametric tests when appropriate,were used (SPSS® software). Since our study was based on amultiparametric approach, we also performed PCA to search for possiblecorrelations between the different parameters that were analyzed(Simca-P® software).

REFERENCES

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Example 2: Triheptanoin Therapy of HD Mouse Models

Aims of the Study

A pilot study is conducted to test the effect of dietary triheptanointherapy versus control diet on selected strains of HD R6/2 mice(Mangariani 1996, Cell 87: 493-506, Kosinski 1999, Neuroreport 10:3891-6). The study includes (i) measuring rates of cerebral anaplerosisfrom heptanoate and brain ATP in R6/2 mice of different ages and incontrol mice in order to demonstrate the ability of triheptanoinmetabolites to cross the blood brain barrier of R6/2 mice and to reversecentral energy deficit; (ii) assessing the therapeutic efficacy oftriheptanoin by accurate behavioral testing, in vivo brain microdialysis(to assay neurotransmitters, triheptanoin metabolites, and BCAA) andneuropathological examination: (iii) metabolomic analyses on mouseplasma and urine.

Research Methods

R6/2 and control mice, on triheptanoin-enriched and control diets, areinfused sequentially—at 4, 8 and 12 weeks—with various doses of [5, 6,7-¹³C₃] heptanoate (by gavage or by intravenous infusion) for 1 hrbefore brain sampling, in order to follow the kinetics of anaplerosis inthe brain (using the assay of anaplerotic CoA esters). Theconcentrations of ATP, ADP, and AMP are assayed in all brain samples, aswell as in muscle of R6/2 mice. We are also assessing the concentrationand labeling pattern of neurotransmitters generated by anaplerosis(GABA, glutamate) to determine whether the brain is deficient in thesecompounds and whether anaplerotic therapy improves these parameters.Brain tissues are be stored at −70° C. in view of possible additionalanalyses (like measurement of oxidative stress).

Behavioral analyses include (i) open field activity monitoring using theTruScan system at 4, 8 and 12 weeks; (ii) RotaRod analysis performedusing the AccuScan system equipped with a shockable door at 6 and 12weeks; (iii) and the Morris Water Maze, the most popular task isbehavioral neuroscience used to assess spatial learning and memory at 12weeks. Other primary endpoints from the study are weight loss andsurvival.

As both HD patients and genetic mouse models of HD manifest apresymptomatic loss of DA receptors, a dysfunctional dopaminergicneurotransmission may be involved in early HD presentation. More recentstudies have shown that DA release is severely compromised in R6/2 mice(Johnson 2006, J Neurochem 97: 737-46). Accordingly, a first-group ofanimals is sacrificed at 12 weeks by cervical dislocation forneurotransmitters analyses. Blood are collected and brain tissue arerapidly removed and dissected on ice into the following regions:striatum, hippocampus, frontal cortex, posterior cortex, cerebellum, andmidbrain. These regions are dissected for both left and righthemispheres of the brain. All right side regions are processed foranalysis of DA, 5-hydroxytryptophane (5-HT) and norepinephrine (NE) andrelated metabolites (3-dihydrophenylacetic acid, homovalinic acid,3-methoxytyramine and 5-hydroxyindolacetic acid) by HPLC withelectrochemical detection. Acetylcholine (Ach) is also measured infrontal posterior cortex tissue by HPLC with electrochemical detection.Branched chain amino acids is measured by HPLC and triheptanoinmetabolites by mass spectrometry. Neuropathology, and especially nuclearneuronal inclusions, is performed on the left side regions. At 12 weeks,a second group of animals is prepared for in vivo microdialysis tomonitor in-vivo release of DA, 5-HT by potassium and Ach release byscopolamine in the striatum. Changes in the extracellular concentrationsof DA, 5-HT and Ach is compared by three ways ANOVA (time×genotype×diet)with repeated measures. Sequential analyses of plasma and urine samplesfrom R6/2 and control mice at 4, 8 and 12 weeks are also perfumed withboth NMR spectroscopy and mass spectrometry. This aims the detection oftriheptanoin metabolites (propionyl and pentanoyl-CoA derivatives) inbody fluids from treated mice. The comparison of the metabolic profilefrom R6/2 and control mice on control diet, by multivariate datastatistical analyses, can possibly confirm the implication of BCAA inthe pathophysiology of R6/2 mice, as evidenced in HD premanifestcarriers and patients, and partially suggested in previous metabolomicstudy in R6/2 mice (Underwood 2006, Brain 129: 877-8). More importantly,the comparison of the metabolic profile from treated versus non-treatedR6/2 mice can assess whether triheptanoin can lead to the correction ofsuch hypercatabolic profiles.

Example 3: Therapy of Spinocerebellar Ataxia 7 (SCA7)

1/In Vitro Trial

To create a simplified model of SCA7 in vitro we used primary culturesof dissociated cerebellar cells because lesion of the cerebellum,particularly the Purkinje cell (PC) layer, accounts for the ataxiaphenotype in patients with SCA7. Our cerebellar cell cultures werecomposed of glial cells and neurons, 5 to 10% of which expressedcalbindin (CaBP) identifying them as PC. To examine the effects ofmutant ATXNT7 on PC survival, the cells were infected at DIV1 (1st DayIn Vitro) with lentiviral vectors carrying truncated formed of normaland mutant ataxin 7 (ATXN7T: amino acids 1-232) fused to GFP(ATXN7T-10Q-GFP, ATXN7T-100Q-GFP). These lentiviral vectors allowedefficient expression of these proteins in about 90% of neurons,including Purkinje cells. Infection by ATXN7T-100Q-GFP led to massiveneuronal loss, almost exclusively in Purkinje neurons (˜85% of Purkinjecell death versus ˜20% loss of other neurons), thus reproducing one ofthe major features of the human disease (FIG. 6).

This model is used to assess the ability of anaploretic molecules torescue Purkinje cells infected by ATXN7T-100Q-GFP. Two compounds aretested: the 3-ketovalerate and the 3-hydroxyvalerate, which are both bedirectly incorporated by the cells in culture. These molecules are addedin the culture medium on the same day when the cells are infected andhalf of the medium is replaced every 4 days. The cultures are maintainedfor 20 days and the potential rescue of Purkinje cells is quantified asdescribed above.

2/In Vivo Experiments

We chose to use the SCA7 Knock-in mouse model developed in the group ofH. Y. Zoghbi (Yoo 2003 Neuron, 37: 383-401), which expresses ATXN7 with266 glutamines at endogenous levels in the proper spatio-temporalpattern. Mouse Sca7 is highly homologous to human SCA7, with 88.7%identity at the protein level. Sca7^(266Q/SQ) mice reproduce features ofinfantile SCA7, which is characterized by a more rapid progression and abroader spectrum of phenotypes than the adult-onset disease. From 3weeks of age, these mice develop progressive weight loss, ptosis, visualimpairment, tremor, ataxia, muscle wasting, kyphosis and finally die ataround 14-19 weeks of age. Sca7^(266Q/SQ) mice manifested coordinationimpairment in the rotarod test by 5 weeks. By 8-9 weeks, gait ataxia isapparent and motor coordination further deteriorates. As in patients,neuropathological studies revealed progressive Nls formation in manybrain regions. Although no neuronal loss is observed in the brain,Purkinje cells that are one of the most commonly affected cells in SCA7,have a decreased body cell size.

a—Metabolic Study of the SCA7 Knock-in Mice

Similarly to patients with HD or SCA7, the SCA7 knock-in mice show asevere progressive weight loss already significant at the onset of themotor phenotype. A protocol has been set up to measure food and beverageintake in correlation with weight evolution of these mutant micecompared to wild-type ones. This protocol is tested on a group of 3knock-in females and 3 wild-type females from the same litters. Theanimals are kept one per cage and they are given, a definite amount offood and beverage. Then, their intake and their weight are measured fourtimes a week. Preliminary results show that this procedure is efficientto evidence progressive weight loss and food intake evolution (FIG. 7).Soon after onset (7-8 weeks) but before serious motor deterioration (8-9weeks) food intake from mutant animals is higher than in wild-typealthough there are already lighter. These data are in favour of thehypothesis of a hypermetabolic state during the early phases of thephenotype. After 8-9 weeks, the locomotor impairment is already sodisabling that the mutant mice probably can't reach the food as easilyas the wild-type ones.

b—Microarray Analysis of Early Transcriptional Modifications

Metabolic impairment in HD and other related diseases have been proposedto result from dysregulation of major metabolic pathways at thetranscriptional level (Mochel 2007 PLoS ONE, 2(7): e647; Cui 2006 Cell127: 59-69). Considering the function of the ATXN7 protein and the earlytranscriptional abnormalities previously evidenced in SCA7 and otherpolyglutamine disease, the transcriptome of the cerebellum of 4-5knock-in mice versus 4-5 wild-type mice is analysed at two early stagesbefore onset (post-natal day 10 and post-natal day 22) and one latesymptomatic stage (11 weeks of age).

Example 4: Evaluation of the Potential Benefit and Safety of AnapleroticTherapy in Huntington Disease (HD)

A 5-days preclinical trial with triheptanoin is 6 HD affected patientsis conducted. This short-term protocol is as follows:

1. Day 1: (i) an extended neurological and general clinical examination;(ii) a global metabolic workup (blood and urine samples) to have anoverview of the metabolic profile of HD patients at baseline; (iii) askin biopsy to test in vitro the ability of triheptanoin to generateenergy from the Krebs cycle and the respiratory chain; (iv) themeasurement of 5′ AMP-activated protein kinase (5′ AMPK) activity inpatients' fibroblasts, as a reflection of the levels of intracellularenergy metabolism; and (v) a ³¹P-NMR spectroscopy on patients' muscle inorder to assess their skeletal muscle ATP production.

2. Day 2: an oral loading test of a meal enriched with, triheptanoin,together with urine and blood samples before and after meal todetermine:

-   -   measurements of triheptanoin metabolites, through plasma        acylcarntines profile and urine organic acids (Roe et al. 2002),        to ensure that triheptanoin is properly metabolized in HD        patients;    -   analyses of mitochondrial redox status, through the ratio of        lactate to pyruvate and 3-hydroxybutyrate to acetoacetate        (Mochel et al. 2005), to assess in vivo the ability of        triheptanoin to generate energy from the Krebs cycle without        overloading the respirator chairs.

3. On days 3, 4 and 5: the pursuit of a diet enriched with triheptanointo determine if a protein sparing effect occurs, i.e. the normalizationof the plasma branched chain amino acids (BCAA) and serum IGF1 levels,and/or the elevation of urinary urea. Clinical examination attempts toidentify acute effects on the systemic energy deficiency (musclestrength, motor function) associated with HD. In addition, patientsundergo a second muscle ³¹P-NMR spectroscopy in order to evidence apossible short-term effect of triheptanoin on patients' peripheralenergy metabolism.

Study Design

On the first day of admission, HD patients are examined. Motordysfunction is evaluated with the Unified Huntington disease ratingscale, UHDRS (Siesling et al. 1998), and a total functional capacityscore, TFC (Marder et al. 2000). General health condition is alsorecorded. In particular history of dysfunction of the digestive tract.Before lunch, blood and urine samples are collected. Standard analysesare performed (blood cell count, blood clotting factors, blood andurine, glucose, serum electrolytes), as well as a global metabolicworkup including plasma redox status (lactate, pyruvate, acetoacetate,3-hydroxybutyrate), plasma amino acids and acylcarnitines, and urineorganic acids as described (Mochel et al. 2005). In the absence of bloodclotting dysfunction, a skin biopsy is performed. A simple functionaltest using propionate labelled with C¹⁴ is further performed in culturedfibroblasts (Benoist et al. 2001). Propionate is one of the mainanaplerotic products of triheptanoin, and is incorporated into proteinproviding that enough ATP is produced from the Krebs cycle and therespiratory chain. The normal rate of protein synthesis, afterincorporation of C¹⁴-propionate, in HD cells therefore reflect theintegrity of the respiratory chain in HD, as well as the possibility togenerate energy from the Krebs cycle through the anaplerotic pathway.The activity of 5′-AMPK, which senses changes in the cellular energystate, is also determined in patients' fibroblasts (Chou et al. 2005),in order to evidence a peripheral deficit in intracellular energymetabolism. In addition, oxidative mitochondrial metabolism isspecifically assessed by muscle ³¹P-NMR spectroscopy using datacollected at the end of a given exercise and daring the followingrecovery (Lodi et al. 2000).

On the second day, HD patients ingest a loading dose of triheptanoin (1g/Kg). For convenience, and better digestive tolerance, triheptanoin isusually administrated together with a dairy product. Repeated bloodsamples are collected before and sequentially, after meal (30, 60, 90,120 and 180 minutes after triheptanoin ingestion) for assessment ofredox status and acylcarnitines profile. Urine is also collected beforeand after the triheptanoin load (90 and 180 minutes) for analyses oforganic acids.

On the next 3 following days, HD patients pursue an isocaloric dietenriched with triheptanoin 1 g/Kg/day divided in 3 to 4 meals). Fastingplasma BCAA, serum IGF1 and urinary urea are analyzed daily andneurological examination is repeated with UHDRS and TFC scoring. On day5, muscle ³¹P-NMR spectroscopy is repeated in order to determine therelative concentrations of inorganic phosphate, phosphocreatine and ATPlevels alter triheptanoin administration.

Patient Selection Criteria

This study involves 6 patients with abnormal CAG repeats expansions(>36) in the HD1 gene, with regular medical and psychological follow-up.The selection of patients is based on:

-   -   UHDRS score ranging 15 and 50, corresponding to patients at an        early to moderate stage of the disease, in order to facilitate        the compliance of patients to dictate treatment;    -   low levels of plasma BCAA, in order to search for a raise in        these amino acids under triheptanoin treatment.

Informed consent is obtained for all participants.

REFERENCES

-   Benoist J F, Acquaviva C, Callebaut I, et al. (2001). “Molecular and    structural analysis of two novel mutations in a patient with mut(−)    methylmalonyl-CoA deficiency.” Mol Genet Metab 72 (2): 181-4.-   Chou S Y, Lee Y C, Chen H M, et al. (2005). “CGS21680 attenuates    symptoms of Huntington's disease in a transgenic mouse model” J    Neurochem 93 (2): 310-20.-   Lodi R, Schapira A H, Manners D, Styles P, Wood N W, Taylor D J and    Warner T T (2000), “Abnormal in vivo skeletal muscle energy    metabolism in Huntington's disease and dentatorubropallidoluysian    atrophy.” Ann Neurol 48 (1): 72-6.-   Marder K, Zhao H, Myers R H et al. (2000). “Rate of functional    decline in Huntington's disease. Huntington Study Group.” Neurology    54 (2): 452-8.-   Mochel F, DeLonlay P, Touati G, et al (2005). “Pyruvate carboxylase    deficiency: clinical and biochemical response to anaplerotic diet    therapy.” Mol Genet Metab 84 (4): 305-12.-   Roe C R, Sweetman L, Roe D S, David F and Brunengraber H (2002).    “Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat    oxidation disorders using an anaplerotic odd-chain triglyceride.” J    Clin invest 110 (2): 259-69.-   Siesling S. van Vugt J P, Zwinderman K A, Kieburtz K and Roos R A    (1998). “Unified Huntington's disease rating scale: a follow up.”    Mov Disord 13 (6): 915-9.

Throughout this application, various references describe the state ofthe art to which tins invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

The invention claimed is:
 1. A method of treating Huntington's disease,the method comprising administering to a patient in need of suchtreatment a composition comprising triheptanoin.
 2. The method of claim1, wherein the triheptanoin is administered orally.
 3. The method ofclaim 1, wherein the triheptanoin is administered parenterally.
 4. Themethod of claim 1, wherein the triheptanoin is administeredintraperitoneally.
 5. The method of claim 1, wherein the compositioncomprises a pharmaceutically-acceptable carrier.
 6. The method accordingto claim 1, wherein the patient has been identified as havingsignificantly lower plasma levels of at least one branched chain aminoacid (BCAA) as compared to a control.
 7. The method according to claim6, wherein the BCAA is selected from valine, leucine, and isoleucine. 8.A method of treating spinal and bulbar muscular atrophy (SBMA), themethod comprising administering to a patient in need of such treatment acomposition comprising triheptanoin.
 9. The method of claim 8, whereinthe triheptanoin is administered orally.
 10. The method of claim 8,wherein the triheptanoin is administered parenterally.
 11. The method ofclaim 8, wherein the triheptanoin is administered intraperitoneally. 12.The method of claim 8, wherein the composition comprises apharmaceutically-acceptable carrier.